Interactive effects of ambient acidity and salinity on thyroid function during acidic and post-acidic acclimation of air-breathing fish (Anabas testudineus Bloch)

The interactive effects of ambient acidity and salinity on thyroid function are less understood in fish particularly in air-breathing fish. We, therefore, examined the thyroid function particularly the osmotic and metabolic competences of freshwater (FW) and salinity-adapted (SA; 20 ppt) air-breathing fish (Anabas testudineus) during acidic and post-acidic acclimation, i.e., during the exposure of fish to either acidified water (pH 4.2 and 5.2) for 48 h or clean water for 96 h after pre-exposure. A substantial rise in plasma T₄ occurred after acidic exposure of both FW and SA fish. Similarly, increased plasma T₃ and T₄ were found in FW fish kept for post-acidic acclimation and these suggest an involvement of THs in short-term acidic and post-acidic acclimation. Water acidification produced significant hyperglycaemia and hyperuremia in FW fish but not in SA fish. The SA fish when kept for post-acclimation, however, produced a significant hypouremia. In both FW and SA fish, gill Na⁺, K⁺-ATPase activity decreased but kidney Na⁺, K⁺-ATPase activity increased upon acidic acclimation. During post-acidic acclimation, gill Na⁺, K⁺-ATPase activity of the FW fish showed a rise while decreasing its activity in the SA fish. Similarly, post-acidic acclimation reduced the Na⁺, K⁺-ATPase activity of intestine but elevated its activity in the liver of SA fish. A higher tolerance of the SA fish to water acidification was evident in these fish as they showed tight plasma and tissue mineral status due to the ability of this fish to counteract the ion loss. In contrast, FW fish showed more sensitivity to water acidification as they loose more ions in that medium. The positive correlations of plasma THs with many tested metabolic and hydromineral indices of both FW and SA fish and also with water pH further confirm the involvement of THs in acidic and post-acidic acclimation in these fish. We conclude that thyroid function of this fish is more sensitive to environmental acidity than ambient salinity and salinity interference nullifies the toxic effect of water acidification.     

Cortisol promotes and integrates the osmotic competence of the organs in North African catfish (Clarias gariepinus Burchell): Evidence from in vivo and in situ approaches

The short-term in situ and long-term in vivo effects of cortisol were examined in North African catfish (Clarias gariepinus) to identify how this major corticosteroid integrates the osmotic competence of fish organs. In the in situ approach, the hydromineral effects of cortisol perfusion (75-300 ng ml⁻¹) for 20 min were tested and the indices of hydromineral and metabolic regulations were measured in our in vivo experimental fish after three alternate intraperitoneal cortisol injections (40 and 200 ng g⁻¹ body mass) for 5 days. Na⁺, K⁺-ATPase activity, a measure of cellular osmotic competence, responded to in situ and in vivo cortisol treatments. In situ cortisol delivery increased the Na⁺, K⁺-ATPase activity in the gill (P < 0.001) and kidney (P < 0.001) but decreased (P < 0.01) in the liver and showed no effect on intestine. In vivo cortisol treatment, on the contrary, increased Na⁺, K⁺-ATPase activity in the gills (P < 0.01), intestine (P < 0.05) and liver (P < 0.01) but decreased (P < 0.05) in the kidney. As expected, plasma cortisol increased (P < 0.001) with increasing doses of cortisol injections which produced direct effects on the metabolites and the mineral contents including the elevations of glucose (P < 0.05), lactate (P < 0.05) and Mg²⁺ (P < 0.05) and reductions of urea (P < 0.05), Na⁺ (P < 0.05) and K⁺ (P < 0.05) in the plasma. A decline of triiodothyronine (P < 0.01) occurred in the catfish after in vivo cortisol treatment and that implies a direct cortisol action on the homeostatic integration in this fish. Evidence is thus presented that in catfish cortisol regulates the whole body hydromineral and metabolite homeostasis by promoting and integrating the osmotic and metabolic functions of the multiple organ systems including liver.     

PRL and GH synthesis and release from the sea bream (Sparus auratus L.) pituitary gland in vitro in response to osmotic challenge

The endocrine factors prolactin (PRL) and growth hormone (GH) are believed to have counteracting effects in the adaption of fish to changes in environmental salinity. In order to further investigate this interaction sea bream were challenged with full seawater (SW) or freshwater (FW) for 7 days and the response of pituitary glands cultured in vitro to an osmotic challenge (230, 275 and 320 mOsm/kg) was assessed. In vitro PRL secretion from pituitaries of SW-adapted fish was unaltered in response to an osmotic challenge, while GH secretion increased in the lowest osmolality (230 mOsm/kg). In contrast, both GH and PRL secretion by pituitaries from FW challenged fish was significantly increased (p < 0.01) over that of pituitaries from SW fish at the highest osmolality (320 mOsm/kg). After FW challenge pituitary PRL content and de novo synthesised and released PRL were significantly increased (p < 0.01), while total PRL secretion was not different from SW animals. GH pituitary content decreased in FW animals while total secretion and secretion of de novo synthesised protein were significantly increased (p < 0.01). In addition, after transfer of fish to FW expression of PRL and GH increased 3- and 2-fold, respectively. Despite the increase in PRL expression, no increase in total PRL secretion occurred and although in gills a 2-fold increase in the osmoregulatory marker, Na⁺/K⁺-ATPase activity was detected, profound haemodilution and a cumulative mortality of 40% occurred in sea bream placed in FW. Taken together the results suggest that the sea bream pituitary gland fails to respond appropriately to the osmotic challenge caused by low salinity and the physiological response evoked in vivo is not enough to allow this species to withstand and adapt to FW.     

Effects of short-term acid and aluminum exposure on the parr-smolt transformation in Atlantic salmon (Salmo salar): disruption of seawater tolerance and endocrine status

Episodic acidification resulting in increased acidity and inorganic aluminum (Al_i) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7–6.9) or acid/Al (pH 5.4–6.3, 28–64 μg l⁻¹ Al_i) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al_i. There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24 h SW challenge exhibited greater plasma Cl⁻ levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na⁺,K⁺-ATPase (NKA) activity and Na⁺,K⁺,2Cl⁻ (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3′,5′-triiodo-l-thyronine (T₃) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T₄) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24 h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T₃.     

Ventricular K currents are reduced in mice with elevated levels of serum TNFα

In the present study mice were treated with tumor necrosis factor alpha (TNFα) for 6 weeks to determine if chronic TNFα treatment could produce serum levels of TNFα similar to what has been observed in disease states (heart failure, HIV) and to determine if these levels of TNFα alter ventricular K⁺ currents. Mice chronically treated with TNFα and sham treated mice were utilized for experiments. Serum levels were measured with a Searchlight® protein array. Patch-clamp techniques, real-time PCR and Western blot analysis were used to study K⁺ current densities and K⁺ channel expression. Results showed that serum concentrations of TNFα were significantly higher in TNFα treated mice compared to controls (control: 9.5 ± 1.5 pg/ml, TNFα: 27.4 ± 5.0 pg/ml; p < 0.05) and comparable to serum TNFα levels observed in heart failure and HIV models. In ventricular myocytes from TNFα treated mice the outward K⁺ currents I_to and I_Kur were significantly reduced (at + 30 mV: I_to: control: 45.0 ± 2.9 pA/pF, TNFα: 34.5 ± 2.9 pA/pF; p < 0.05; I_Kur: control 34.1 ± 2.7 pA/pF, TNFα: 25.0 ± 2.2 pA/pF; p < 0.05). Expression studies revealed that ventricular mRNA and protein expression for the channels underlying I_to and I_Kur did not differ between the two groups. However, the recovery from inactivation for I_Kur was significantly longer in TNFα treated mice. Overall, this study shows that pathologically relevant levels of serum TNFα modulate K⁺ currents in mouse ventricle. These findings could help to explain the role of TNFα in the pathogenesis of cardiac arrhythmia.     

Estrogen exerts concentration-dependent pro-and anti-hypertrophic effects on adult cultured ventricular myocytes. Role of NHE-1 in estrogen-induced hypertrophy

Estrogen has been shown to protect the heart and attenuate myocardial hypertrophy and left ventricular remodelling through as yet to be defined mechanisms. In the present study we examined concentration-dependent effects of estrogen on hypertrophy of adult rat cardiomyocytes, potential underlying mechanisms related to intracellular pH (pH_i) and possible sex-dependent responses. Cardiomyocytes were isolated from adult male and female Sprague-Dawley rats and used immediately for pH_i determinations or cultured and subsequently treated for 24 h with 17β-estradiol to assess hypertrophic responses. Fluorometric measurements with the pH_i-sensitive dye BCECF demonstrated that at 1 pM 17β-estradiol increased pH_i (+ 0.05 pH units in females and + 0.12 pH units in males, P < 0.05) by a rapid non-genomic mechanism that was blocked by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 μM). Treatment with 1 pM 17β-estradiol for 24 h increased cell size (females: 20%, P < 0.05; males: 29%, P < 0.05) and ANP expression (females: 414%, P < 0.05; males: 497%, P < 0.05) in a NHE-1-, and ERK1/2 MAPK-dependent manner. At 1 nM, 17β-estradiol decreased pH_i (females: − 0.24 pH units, P < 0.05; males: − 0.07 pH units, P < 0.05) which was also prevented by AVE, although at this concentration the hormone had no direct hypertrophic effect but instead prevented hypertrophy induced by phenylephrine. Our results show that low levels of estrogen produce cardiomyocyte hypertrophy through ERK/NHE-1 activation and intracellular alkalinization whereas an antihypertrophic effect is seen at high concentrations. These effects may further our understanding of the role of estrogen in heart disease particularly associated with hypertrophy.     

Ca/calmodulin-dependent protein kinase II contributes to intracellular pH recovery from acidosis via Na/H exchanger activation

The Na⁺/H⁺ exchanger (NHE-1) plays a key role in pH_i recovery from acidosis and is regulated by pH_i and the ERK1/2-dependent phosphorylation pathway. Since acidosis increases the activity of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in cardiac muscle, we examined whether CaMKII activates the exchanger by using pharmacological tools and highly specific genetic approaches. Adult rat cardiomyocytes, loaded with the pH_i indicator SNARF-1/AM were subjected to different protocols of intracellular acidosis. The rate of pH_i recovery from the acid load (dpH_i/dt)—an index of NHE-1 activity in HEPES buffer or in NaHCO₃ buffer in the presence of inhibition of anion transporters—was significantly decreased by the CaMKII inhibitors KN-93 or AIP. pH_i recovery from acidosis was faster in CaMKII-overexpressing myocytes than in overexpressing β-galactosidase myocytes (dpH_i/dt: 0.195 ± 0.04 vs. 0.045 ± 0.010 min^− 1, respectively, n = 8) and slower in myocytes from transgenic mice with chronic cardiac CaMKII inhibition (AC3-I) than in controls (AC3-C). Inhibition of CaMKII and/or ERK1/2 indicated that stimulation of NHE-1 by CaMKII was independent of and additive to the ERK1/2 cascade. In vitro studies with fusion proteins containing wild-type or mutated (Ser/Ala) versions of the C-terminal domain of NHE-1 indicate that CaMKII phosphorylates NHE-1 at residues other than the canonical phosphorylation sites for the kinase (Ser648, Ser703, and Ser796). These results provide new mechanistic insights and unequivocally demonstrate a role of the already multifunctional CaMKII on the regulation of the NHE-1 activity. They also prove clinically important in multiple disorders which, like ischemia/reperfusion injury or hypertrophy, are associated with increased NHE-1 and CaMKII.     

Corticosteroid regulation of Na,K-ATPase α1-isoform expression in Atlantic salmon gill during smolt development

The proposed mineralocorticoid-like signalling axis in teleost fish, consisting of the hormone 11-deoxycorticosterone (DOC) and a mineralocorticoid receptor (MR), has recently challenged our conception of cortisol being the only osmoregulatory corticosteroid in teleost fish. This paper aimed at comparing the osmoregulatory role of DOC with that of cortisol during the pre-adaptive development of SW-tolerance, smoltification, in Atlantic salmon. Using an in vitro gill block incubation system, the effect of DOC and cortisol in the gill was investigated from January to September, using Na⁺,K⁺-ATPase α-subunit isoforms α-1a and α-1b mRNA levels as targets for regulation by the hormones. Cortisol and DOC both conferred significant up-regulation of α-1a and α-1b mRNA levels at specific time-points during smoltification. However, the effect of cortisol and DOC on α-subunit isoforms varied seasonally between isoforms and hormones. The maximum induction of α-1a was 3- to 4-fold compared to controls whereas a 2-fold induction was observed for α-1b. The pattern and capacity of stimulation of α-1a through smoltification were similar for cortisol and DOC, whereas cortisol had an enhanced capacity to stimulate α-1b as compared to DOC. Even though there was no demonstrable change in cortisol or DOC sensitivity in the gill, the magnitude of the hormonal effects were seasonally dependent. This is the first report of DOC-induced effects on osmoregulatory targets in fish, thus indicating a role for DOC and MR signalling in osmoregulation.     

Normal passive viscoelasticity but abnormal myofibrillar force generation in human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, increased ventricular stiffness and impaired diastolic filling. We investigated to what extent myocardial functional defects can be explained by alterations in the passive and active properties of human cardiac myofibrils. Skinned ventricular myocytes were prepared from patients with obstructive HCM (two patients with MYBPC3 mutations, one with a MYH7 mutation, and three with no mutation in either gene) and from four donors. Passive stiffness, viscous properties, and titin isoform expression were similar in HCM myocytes and donor myocytes. Maximal Ca²⁺-activated force was much lower in HCM myocytes (14 ± 1 kN/m²) than in donor myocytes (23 ± 3 kN/m²; P < 0.01), though cross-bridge kinetics (k_tr) during maximal Ca²⁺ activation were 10% faster in HCM myocytes. Myofibrillar Ca²⁺ sensitivity in HCM myocytes (pCa₅₀ = 6.40 ± 0.05) was higher than for donor myocytes (pCa₅₀ = 6.09 ± 0.02; P < 0.001) and was associated with reduced phosphorylation of troponin-I (ser-23/24) and MyBP-C (ser-282) in HCM myocytes. These characteristics were common to all six HCM patients and may therefore represent a secondary consequence of the known and unknown underlying genetic variants. Some HCM patients did however exhibit an altered relationship between force and cross-bridge kinetics at submaximal Ca²⁺ concentrations, which may reflect the primary mutation. We conclude that the passive viscoelastic properties of the myocytes are unlikely to account for the increased stiffness of the HCM ventricle. However, the low maximum Ca²⁺-activated force and high Ca²⁺ sensitivity of the myofilaments are likely to contribute substantially to any systolic and diastolic dysfunction, respectively, in hearts of HCM patients.     

Changes in gene expression levels of somatolactin in the pituitary and morphology of gill mitochondria-rich cells in Mozambique tilapia after transfer to acidic freshwater (pH 3.5)

Mozambique tilapia, Oreochromis mossambicus, is easily acclimated to highly acidic water, and thus presents a useful model to unravel endocrine regulation of adaptation to acidic water in fish. We analyzed gene expression of somatolactin (sl), growth hormone (gh) and prolactin (prl), in the pituitary gland and size distribution of mitochondria-rich (MR) cells in the gills after transfer from normal freshwater (FW, pH 7.2) to acidified freshwater (AW, pH 3.5). Plasma osmolality drastically decreased until 2 days after transfer to AW, but had restored to normal after 1 week of acclimation, and this confirmed the excellent acid tolerance of tilapia. Expression levels of sl, gh and prl were all up-regulated during short-term exposure to AW. The expression of sl remained elevated up to 7 days after transfer; the expression of gh and prl was back to initial levels at that time. These findings point to an important and specific role of SL in adaptation to acid water in this tilapia, although temporal contribution of GH and PRL cannot be ruled out. The size distribution of branchial MR cells changed drastically during acclimation to AW. The mean MR cell size was 1.5-fold larger in the fish exposed to AW for 7 days compared to controls in FW. The gills and their MR cells are a likely site of important acid-base regulation, and SL may change ion-transport functions of MR cells to correct plasma osmotic balance disturbed by acid exposure.     

Angiotensin II inhibits the electrogenic Na/HCO3 cotransport of cat cardiac myocytes

The Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K⁺ [K⁺]_o from 4.5 to 45 mM (K⁺ pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19 ± 0.008, n = 6; data expressed as an increase of pH_i units after 14 min of applying the K⁺ pulse). This alkalization was completely abrogated by the NBC blocker S0859 (− 0.004 ± 0.016*, n = 5; * indicates p < 0.05 vs control) but not by the Na⁺/H⁺ exchanger blocker HOE642 (0.185 ± 0.04, n = 4), indicating that we are exclusively measuring eNBC. The K⁺ pulse induced alkalization was canceled by 100 nM Ang II (− 0.008 ± 0.018*; n = 5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT₁ receptor blocker, 0.18 ± 0.02; n = 4) or SB202190 (p38 MAP kinase inhibitor, 0.25 ± 0.06; n = 5). Neither chelerythrine (PKC inhibitor, − 0.06 ± 0.04*; n = 4), nor U0126 (ERK inhibitor, − 0.07 ± 0.04*; n = 4) nor MPG (ROS scavenger, − 0.02 ± 0.05*; n = 8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT₁ receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.     

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p < 0.05) in the presence of hydrogen peroxide (H₂O₂). No significant differences (p > 0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p < 0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 × 10⁻¹² mol crab⁻¹), the daily variation in TPRSC values was abolished (p > 0.05). This molecule did not change the NP-SH content (p > 0.05) in vitro, but decreased (p < 0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p < 0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.     

Cardiovascular risk in obesity: Different activation of inflammation and immune system between obese and morbidly obese subjects

Background

Both inflammation and immunity are involved in the development and progression of atherosclerosis. Obesity is considered a major modifiable cardiovascular risk factor, however, the correlation between increasing degrees of obesity and cardiovascular risk is not clear yet. Aim of our study was to investigate how different degrees of obesity are associated with inflammation and immune system responses.

Methods

One-hundred healthy individuals were divided into 3 groups according to body mass index (BMI): 22 overweight (OW), 26 obese (O) and 52 morbidly obese (MO). High-sensitivity C-Reactive Protein (hs-CRP, immunonephelometry), leptin (radio-immunoassay) and CD4⁺CD28^nullT-lymphocytes (flow-cytometry), a particular subset of T-lymphocytes with pro-atherogenic and plaque-destabilizing properties, were assessed.

Results

hs-CRP levels were significantly higher in O vs OW (p = 0.036), in MO vs OW (p < 0.001) and in MO vs O (p = 0.012). Similarly, leptin levels were higher in O vs OW (p = 0.02), in MO vs OW (p < 0.001) and in MO vs O (p < 0.001). CD4⁺CD28^nullT-lymphocytes were higher in O vs OW (p < 0.001), in O vs MO (p = 0.03) and in MO vs OW (p = 0.01). hs-CRP and leptin levels significantly correlated each other (r = 0.39; p < 0.001) and with waist circumference (r = 0.52; p < 0.001; r = 0.64; p < 0.001) and BMI (r = 0.60; p < 0.001; r = 0.74; p < 0.001).

Conclusions

Our study demonstrates that, notwithstanding higher levels of inflammation, MO are characterized by less detrimental immune activation, as shown by the reduced CD4⁺CD28^nullT-cells expansion as compared to OW and O, which might translate in less immune vascular injury. These findings suggest that MO might represent a particular population, in which different pathophysiological mechanisms take part if compared with “classic” obesity.     

Reduced SERCA2a converts sub-lethal myocardial injury to infarction and affects postischemic functional recovery

The goal of the present study was to assess how reduced SERCA2a expression affects in vivo myocardial ischemia/reperfusion (I/R) injury. We specifically wanted to determine to what extent hearts with reduced SERCA2a levels are susceptible to in vivo I/R injury. Therefore, we examined the effects of different ischemic periods on post-ischemic myocardial injury in wild-type (WT) and SERCA2a heterozygous knockout (SERCA2a^+/−) mice expressing lower levels of SERCA2a pump in vivo. Following 20-min ischemia and 48-hour reperfusion, SERCA2a^+/− mice developed significant myocardial infarction (MI) compared to negligible infarction in WT mice (14 ± 3% vs. 3 ± 1%, P < 0.01); whereas following 30-min ischemia, the infarction was significantly larger in SERCA2a^+/− mice compared to WT mice (49 ± 5% vs. 37 ± 3%, P < 0.05). Further, echocardiographic analysis revealed worsened postischemic contractile function in SERCA2a^+/− mice compared to WT mice. Thus, these findings demonstrate that maintaining optimal SERCA2a function is critical for myocardial protection from I/R injury and postischemic functional recovery.     

Transmissibility, by Glossina morsitans morsitans, of Trypanosoma congolense strains during the acute and chronic phases of infection

In order to verify whether chronic trypanosomal infections can affect the transmissibility of Trypanosoma congolense by tsetse flies, batches of Glossina morsitans morsitans were fed on mice infected with the same level of parasitemia (10^8.1 trypanosomes/ml of blood) of two cloned low virulent T. congolense strains during the acute and the chronic phases of infection. Results showed that the proportions of procyclic infections in flies that were fed during the acute phase (32.6% and 45.4% for isolates 1 and 2, respectively) were significantly higher (χ² = 4.7, P < 0.05 and χ² = 23.7, P < 0.0001, respectively) compared to the proportions of procyclic infections of flies fed during the chronic phase of infection (18.8% and 14.9% for isolates 1 and 2, respectively). Similarly the proportions of metacyclic infections in flies fed during the acute phase (32.6% and 45.4% for isolates 1 and 2, respectively) were significantly higher (χ² = 6.3, P < 0.05 and χ² = 23.7, P < 0.0001, respectively) compared to the proportions of metacyclic infections in flies fed during the chronic phase of infection (16.8% and 14.9% for isolates 1 and 2, respectively). No significant difference was found in the maturation rate of both strains during the acute phase compared to the chronic phase of infection (P > 0.05). The results of this study suggest that T. congolense loses part of its transmissiblity by tsetse flies during the chronic phase of infection.     

Selective block of delayed rectifying potassium current in the rabbit sinoatrial node by a novel class III antiarrhythmic agent MS-551

Summary Electrophysiological actions of MS-551, a novel class III antiarrhythmic agent, were studied using small preparations (0.2 × 0.2 × 0.1mm) of the rabbit sinoatrial (SA) node. MS-551 (0.1–3 µM) exerted a negative chronotropic action by prolonging the action potential duration and diastolic interval. Automaticity was completely suppressed in 5 of 6 preparations at 1–3 µM. Voltage clamp experiments using double microelectrode techniques revealed that MS-551 (0.1–10µM) blocked the delayed rectifying K⁺ current (I_K) in a concentration-dependent manner, and the block was almost saturated at >1 µM, attaining 60% ± 10% at 10 µM (n = 5). The MS-551-sensitive I_K tail (K_d = 0.4µM, Hill r = 1.4,n = 5) had fast and slow components of deactivation. MS-551 (1 µM) reduced the amplitudes of control I_K fast and I_K slow from 20 ± 4 and 11 ± 4 nA to 8 ± 3 and 5 ± 3 nA, respectively (P < 0.01,n = 4). Although the fast deactivation time constant at –60mV remained unaltered (127 ± 12 vs 113 ± 13ms), the slow deactivation time constant was prolonged from 1,117 ± 130 to 1,555 ± 407ms by 1µM MS-551 (P < 0.05). This agent shifted the steady-state activation curve for I_K from –21 ± 2 to –26 ± 4mV and increased the slope factor from 8 ± 1 to 9 ± 1mV (P < 0.05,n = 4). The fully-activated I_K exhibited prominent inward rectification and was reduced by MS-551. These results suggest that (1) MS-551 prolongs the action potential duration and diastolic interval, and exerts a negative chronotropic action by blocking I_K, (2) MS-551 has a higher affinity for the activated than the resting state K⁺ channel, and (3) this agent may either preferentially block one type of I_K, or stabilize a single population of I_K in a subconductance state in the rabbit SA node.     

Estrogenic compounds decrease growth hormone receptor abundance and alter osmoregulation in Atlantic salmon

Exposure of Atlantic salmon smolts to estrogenic compounds is shown to compromise several aspects of smolt development. We sought to determine the underlying endocrine mechanisms of estrogen impacts on the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Smolts in freshwater (FW) were either injected 3 times over 10 days with 2 μg g⁻¹ 17β-estradiol (E2) or 150 μg g⁻¹ 4-nonylphenol (NP). Seawater (SW)-acclimated fish received intraperitoneal implants of 30 μg g⁻¹ E2 over two weeks. Treatment with these estrogenic compounds increased hepatosomatic index and total plasma calcium. E2 and NP reduced maximum growth hormone binding by 30-60% in hepatic and branchial membranes in FW and SW, but did not alter the dissociation constant. E2 and NP treatment decreased plasma levels of IGF-I levels in both FW and SW. In FW E2 and NP decreased plasma GH whereas in SW plasma GH increased after E2 treatment. Compared to controls, plasma chloride concentrations of E2-treated fish were decreased 5.5 mM in FW and increased 10.5 mM in SW. There was no effect of NP or E2 on gill sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) activity in FW smolts, whereas E2 treatment in SW reduced gill Na⁺/K⁺-ATPase activity and altered the number and size of ionocytes. Our data indicate that E2 downregulates the GH/IGF-I-axis and SW tolerance which may be part of its normal function for reproduction and movement into FW. We conclude that the mechanism of endocrine disruption of smolt development by NP is in part through alteration of the GH/IGF-I axis via reduced GH receptor abundance.     

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na⁺,K⁺-ATPase activity in the coastal SW fish and the plasma Na⁺ levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.